Electrophoretic separator



July'l4,'1970 A. KOLIN ELECTROPHORETIC SEPARATOR 2 Sheets-Sheet 1IMPROVED COLUMN Filed Sept. 29, 1967 PRIOR ART COLUMN INVENTOR.ALEXANDER KOLI N ERVl/V F JOHNSTON ATTORNEY y 1970 A. KOLIN 3,520,793

ELECTROPHORETIC SEPARA'I'OR Filed Sept. 29, 1967 2 Sheets-Sheet UnitedStates Patent 3,520,793 ELECTROPHORETIC SEPARATOR Alexander Kolin, LosAngeles, Calif., assignor, by mesne assignments, to the United States ofAmerica as represented by the Secretary of the Navy Filed Sept. 29,1967, Ser. No. 671,894 Int. Cl. B01k 5/00 US. Cl. 204-299 6 ClaimsABSTRACT OF THE DISCLOSURE The description discloses an improvement foran electrophoretic separator of the type wherein particles dissolved orsuspended in a fluid are separated by subjecting the fluid to a combinedaction of a longitudinal electric field traversed by a perpendicularmagnetic field within a horizontally extending migration column. Themigration column is an endless fluid belt bounded on the inside by asoft iron core which is spaced from a surrounding jacket which forms theouter boundary of the fluid belt. On opposite ends of the fluid belt aredisposed buffer chamhers which are capable of supplying a buffer mediumfor carrying the particles which are to be separated. This separator,which is fully described in Proceedings of the National Academy ofSciences, vol. 46 at page 509, utilizes a circular migration column asseen in cross section. Thepresent improvement employs a noncircularmigration column, shaped like the belt of a belt sander, whichovercomes, particle sedimentation problems.

The invention described herein may be manufactured and used by or forthe Government of the United States of America for governmental purposeswithout the payment of any royalties thereon or therefor.

The ions within a biological or chemical mixture have different ionicmobilities which enable the ions to be separated when they are movedthrough an electric field. The apparatus for achieving such a result isan electrophoretic separator which has potential use in separatingchemical mixtures or biological mixtures such as blood. For instance,the separation of blood into its components could isolate abnormalcells, cell debris, or virus particles which would be pertinent indiagnosing ailments.

In my patent application entitled Fractionation Apparatus, Ser. No.500,817 and filed Oct. 22, 1965, now Pat. No. 3,451,918, I describe anelectrophoretic separator which has a horizontal migration column whichis circular in vertical cross section. Disposed at opposite ends of themigration column are a pair of chambers for providing a buffer flowwhich will carry a fluid under test through the migration column. Uponapplying radial magnetic and axial electric fields with respect to themigration column, the solution or suspension under test will beseparated into its various components within the column. This separatoris also described in vol. 46 of the Proceedings of the National Academyof Sciences at p. 509.

Because of the difference of densities between the particles of thefluid under test and the carrying buffer solution the particles willtend to sediment by gravity to the .inner and outer walls of themigration column. With a circular migration column, which is used in mypreviously described electrophoretic separator, this problem is quiteacute, as illustrated in FIG. 1. In order to avoid sedimentation in myprevious separator it is necessary to maintain a rapid rate ofrotational flow of the buffer containing the particles under test. Ifound that the sedimentation problems can be overcome by extending thevertical dimension of the migration column, as illustrated in FIG. 2.With such an arrangement the particles spend most of 'ice their time invertical paths, where gravity causes no sedimentation toward a wall, andonly a small fraction of their cycle in semicircular top and bottompaths, where there is a tendency to sediment toward a wall.

An object of the present invention is to provide an im provement of anelectrophoretic separator which is described in my patent applicationentitled Fractionation Apparatus, Ser. No. 500,817, filed Oct. 22, 1965,now Pat. No. 3,451,918.

A further object is to provide an improved electrophoretic separatorwherein sedimentation of particles flowing through the migration columnthereof is minimized.

Other objects, advantages and novel features of the invention willbecome apparent from the following detailed description of the inventionwhen considered in conjunction with the accompanying drawings wherein:

FIG. 1 is a schematic illustration of a cross-section of the migrationcolumn of my previous electrophoretic separator;

FIG. 2 is a schematic illustration of a cross-section of my improvedmigration column;

FIG. 3 is a schematic illustration, partially in crosssection, of anelectrophoretic separator which includes my improved column; and

FIG. 4 is a view taken along plane IVIV of FIG. 3.

Referring now to the drawings wherein like reference numerals designatelike or similar parts throughout the several views, there is shown inFIG. 3 my improved electrophroetic separator 10 which is an improvementof an electrophoretic separator described in my previously mentionedpatent application. The electrophoretic separator 10 has a migrationcolumn 12 which is bounded by an iron core 14 and a surrounding jacket16. The configuration of this column 12, which is illustratedschematically in FIG. 2, provides improved results of the separator overthe configuration of my previous migration column, which isschematically illustrated in FIG. 1. As illustrated in FIG. 3, theimproved migration column 12 extends horizontally and, as illustrated inFIG. 2, the migration column in vertical section has a large verticaldimension in contrast to its transverse dimension.

A fluid under test is injected into the migration column 12 frominjector 68 and is carried through it along helical paths in a buffersolution. This fluid and solution will be described in detailhereinafter. During transition within the migration column the fluidparticles traverse a magnetic field which is perpendicular to thesurface of an iron core 14 and are subjected to a horizontal electricfield which is perpendicular to the magnetic field and which causesseparation of the particles according to their ionic mobilities.

Because of the difference in densities between the fluid particles andthe carrier solution there is a tendency of the particles to migrate bygravity toward the walls of the migration column. FIG. 1, which is theprior art column, illustrates the deviation of a fluid particle which ismore dense than the buffer solution from a center path after theparticle has been discharged into the migration column 18. It can beseen that after injection the more dense fluid particle will, bygravity, first deviate inwardly toward the iron core 22 and will thensubsequently deviate, also by gravity effect, toward the inner wall ofthe jacket 24. This deviation will cause the fluid particles which aremore or less dense than the buffer solution to sedimentate on the wallsof the migration column 18 unless rotational flow of the buffer fluid ismaintained at a high rate. My improved migration column, as illustratedin FIG. 2, has overcome this problem by employing a noncircularconfiguration which causes the fluid particles to spend a greater amountof their time in vertical paths 26 and 28, where gravity has nodeviation effect, in contrast to top and bottom paths 30 and 32, wherethere is a horizontal component. The deviation of the particles in thetop and bottom path portions 30 and 32 is so slight that it is not shownin FIG. 2. As can be seen from FIG. 1, the entire length of the particlepath has a horizontal component which subjects the particles to gravitydeviation.

The preferred configuration of my improved migration column 12, as seenin cross section is generally rectangular with top and bottom roundedends, as illustrated in FIG. 2. It is important that the legs 26 and 28of the particle path be oriented vertically. The rounded top and bottomends 30 and 32 are preferably semicircular, as illustrated. Verysatisfactory results can be achieved by employing an iron core 14 withthe following dimensions; a horizontal length of approximately 7.5 cm.;a height of approximately 10.6 cm.; a thickness of approximately 2.6cm.; and a radius of curvature at its top and bottom ends ofapproximately 1.3 cm.; the first dimension being illustrated in FIG. 3,and the latter three dimensions being illustrated in FIG. 2. The jacket16, which may be constructed of Lucite, may be evenly spacedapproximately 1.5 mm. about the iron core 14 so as to provide themigration column 12 with a uniform width of the same dimension about itsvertical section, as seen in FIG. 2. In order to provide propercentering of the iron core 14 within the jacket 16 small cylindricalspacers (not shown) 1.5 mm. in thickness may be placed around the rim ofthe core 14 between the core and the jacket 16. It is important that theiron core 14 be insulated against electrical contact with thesurrounding buffer solution, which will be described hereinafter. Thisinsulation may be provided by painting the core with a spray varnish orby molding the core 14 into a crust epoxy which is subsequently milledoff to leave a layer of a few thousandths of an inch adhering to thecore 14 over its entire surface. If desired, the epoxy surface may bepainted in suitable colors so that particle streaks may be easily viewedthrough the Lucite jacket 16.

Disposed at opposite vertical ends of the iron core 14 and the migrationcolumn 12 are buffer chambers 34 and 36. The buffer chambers 34 and 36communicate electrically and hydraulically with one another through themigration column 12. Located adjacent the outer ends of the bufferchambers 34 and 36 are electrode compart ments 38 and 40 which may beseparated from the buffer compartments by dialyzing membranes 42 and 44.The dialyzing membranes 42 and 44 hydraulically isolate the buffercompartments from the electrode compartments but enable electricalcommunication therebetween. The buffer compartments 34 and 36 may begenerally rectangular, as seen in vertical cross section (not shown),and the electrode compartments 38 and 40 may be divided into twosections 40a and 40b, as illustrated for the electrode compartment 40 inFIG. 4. These electrode compartment sections may be joined by a conduit46 for hydraulic communication of the buffer solution, and may havebuffer overflow conduits 48 and 50 at their top ends. The buffercompartments 34 and 36 and the electrode compartments 38 and 40 may beformed -by Lucite walls, as illustrated in FIG. 3. The top of the buffercompartments 34 and 36 may be open so as to receive a buffer solution.

The buffer solution, which may be contained in a Mariotte bottle 52, isdistributed to the butler compartments 34 and 36 and the electrodecompartments 38 and 40 through various tubes and conduits. A pluralityof thin plastic tubes divided into bundles 54 and 56 may be utilized fordistributing the buffer solution to the buffer compartments 34 and 36.The apportionment of these tubes among the compartments 34 and 36determines the rate and direction of axial buffer flow in the migrationcolumn 12. In actual practice these tubes 54 and 56 are submerged intothe buffer solution within the buffer compartments 34 and 46. The flowof buffer solution into these compartments may be cut by a stopcock 58which opens into a manifold 60 and may be adjusted by varying the heightof the Mariotte bottle 52. The buffer solution may be distributed fromthe Mariotte bottle 52 to the electrode compartments 38 and 40 through aconduit 62 which has branches opening respectively into the bottoms ofthe electrode compartments. A stopcock 64 may be employed forcontrolling the rate of flow of the buffer solution into the electrodecompartments. The buffer solution may be composed of a pH 10 buffertablet in one liter of water or some other type of solution of suitablepH suitably diluted may be used.

If a greater amount of the buffer solution is distributed to the buffercompartment 34 than the buffer compartment 36, there is a buffer flow tothe left through the migration of column 12, as can be visualized fromFIG. 3. Into this buffer flow is injected the fluid containing particlesto be separated. This test fluid may be disposed within a reservoir andmay be ejected into the migration column 12 near its upstream end by aninjector 68 which is in communication with the reservoir 66 through aconduit 70. Accordingly, the fluid under test will be carried to theleft within the migration column 12 by the buffer solution, as can bevisualized from FIG. 3. During this flow the fluid under test, as wellas the buffer fluid, is subjected to combined magnetic and electricalfields. The magnetic field may be provided by two pairs of bar magnets,one pair 72 and 74 being disposed at the right end of the iron core 14and the other pair 76 and 78 being disposed at the left end of the ironcore. Accordingly, the iron core 14 is sandwiched between the pairs ofmagnets. The pairs of magnets, which may be rectangular bars, aredisposed with their polarities in opposition with respect to oneanother. Accordingly, the north poles of the pair of magnets 72 and 74may be placed in opposition with the north poles of the other pair ofmagnets 76 and 78. If desired the south poles could have been used foropposition purposes. The magnetic field generated corresponds to theradial magnetic field of the circular configuration shown in FIG. 1. Itpenetrates the migration column 12 at right angles to the surface ofiron core 14. The axial electric field through the migration column 12may be provided by pairs of electrodes 80 and 82, one pair of electrodes82a and 82b being disposed respectively within subcompartments 40a and40b of the electrode compartment 40, as illustrated in FIG. 4, and theother pair of electrodes 80 being similarly disposed within theelectrode compartment 38. The pair of electrodes 80 may be negativelycharged and the pair of electrodes 82 may be positively charged by apotential source (not shown).

The pairs of magnets and the electrodes subject the fluid under test aswell as the buffer solution to crossed electric and magnetic fields.This results in a force which makes the fluid in the migration column 12circulate in the manner of motion of an endless belt. As a result ofthis circulation and the axial streaming to the left, the fluidparticles describe a helical path. If the fluid under test has particlesof varying ionic mobilities the electric field will add a horizontalcomponent to their velocity and will separate these particles as theymigrate to the left within the migration column 12 along spiral paths ofdilfering pitch. The fluid particles of differing ionic mobilities willseparate into individual streaks. A particle of slow ionic mobility isillustrated along the solid path 84 of FIG. 3, and a particle of fasterionic mobility is illustrated along the dotted path 86. These paths arehelices of different pitch. The various particles of the fluid undertest are then collected by a bundle of tubes 88 which have one series ofends disposed within the migration column 12 along a parallel to theaxis of the iron core 14, and the opposite series of ends extending fromthe migration column 12 for collection purposes by a series of testtubes 90.

In order to suppress thermal convection of the fluid within themigration column 12 I have found it desirable to minimize the hydrauliccommunication of the buffer solution between the buffer compartments 34and 36, and the migration column 12. This may be accomplished by a pairof nylon shoe laces 92 and 94 which are fitted tightly about respectiveopposite ends of the iron core 14 between the buflfer compartments 34and 36. In order to allow unimpeded replenishment of the fluid leavingvia the collector tubes 88 a small opening 96 may be cut into the leftshoe lace 94 just below the collector tube ends.

In order to eliminate undesirable heating of the separator it} a coolingsystem may be employed for the migration column 12. The iron core 14 maybe hollow so that ice water from a reservoir 98 may be perfusedtherethrough by inlet and outlet conduits 160 and 102. Further, thejacket 16 about the iron core may be provided with a peripheral chamber104 which receives ice water from the reservoir 98 via a conduit 1%. Thereturn from the chamber 104 may be via the conduit 102. In order todispel air from the hollow core 14 when it is first charged with iceWater a conduit 108 and air outlet valve 119 may be connected to the topend of the iron core 14.

It is now readily apparent that the present invention will enable a moreeffective and efficient use of my previously described electrophoreticseparator. By extending the vertical dimension of the migration column12 the n particles to be separated will describe paths which areessentially unaffected by gravity deviation. Accordingly, the improvedseparator will have a much higher resolution and will permit processingof test fluids of a higher concentration. Another advantage is thatfewer turns of the helix are required per given length of path of theinjected particles in the new apparatus than was needed in the originalcircular version.

Obviously many modifications and variations of the present invention arepossible in the light of the above 2 teachings. It is therefore to beunderstood that within the scope of the appended claims the inventionmay be practiced otherwise than as specifically described.

I claim: 1. In an electrophoretic separation apparatus which isstabilized against thermal convection, the combination of:

a base structure; a first tubular member mounted on said base structureand defining an electrophoretic migration column; said electrophoreticcolumn being generally rectangular in vertical cross-section with topand bottom rounded paths and elongated paths therebetween, saidelongated paths extending vertically; a first housing defining a firstbuffer fluid chamber mounted on said base at one end of the said tubular7 member;

a second housing defining a second buffer fluid chamber mounted on saidbase at the other end of said tubular member;

said first tubular member intercoupling said first and second bufferfluid chambers for the flow of fluid through said tubular member fromsaid first chamber;

first and second electrodes respectively positioned in said first andsecond chambers for producing an electric field longitudinally of saidcolumn between said first and second chambers so as to cause a migrationof particle components of a substance to be separated along said column;

magnetic means for producing a magnetic field in said column extendinggenerally radially across said column and traversely to said electricfield to exert tangential forces on said particle components migratingalong said column to cause said particle com ponents to assume differentspiral-like paths;

a source of butter fluid coupled to said first and second butterchambers and having means for continuously supplying buffer fluid tosaid chambers and maintaining an essentially constant level of buflerfluid in said chambers to compensate for extraction losses and tomaintain a predetermined axial flow distribution in said column;

means for injecting into said first tubular member a substance to beelectrophoretically Separated by the buffer fluid flow in said migrationcolumn; and

pick-off means positioned in said migration column to intersect saidparticle components migrating along said spiral paths.

2. In an electrophoretic separator as claimed in claim It wherein:

each of the top and bottom rounded paths is semi circular.

3. In an electrophoretic separator as claimed in claim 2 wherein:

the dimension of the height of the electrophoretic column between itstop and bottom semicircular paths is larger than the transversedimension of width as measured between the centers of the two oppositevertical paths of the column.

4. In an electrophoretic separator as claimed in claim 2 wherein:

the dimension of the height of the electrophoretic column between itstop and bottom semicircular paths is approximately four times thedimension of the width of the column between its vertical elongatedpaths.

5. In an electrophoretic separator as claimed in claim 4 wherein:

said electrophoretic column is formed by an iron core which issurrounded by a jacket; and including:

a thin layer of insulation bonded to said iron core.

6. In an electrophoretic separator as claimed in claim 5 wherein:

the iron core and jacket are both hollow; and including: means forcirculating a coolant through the iron core and the jacket.

References Cited UNITED STATES PATENTS 3,207,684 9/1965 Dotts 204-4803,287,244 11/1966 Mel 204-18O 3,305,471 2/1967 Miinchhausen et al.204299 3,320,148 5/1967 Skeggs 204180 3,451,918 6/1969 Kolin 204299 JOHNH. MACK, Primary Examiner 0 A. C. PRESCOTT, Assistant Examiner US. Cl.X.R. 204180

